Process for the preparation of powdered probiotic formulations for monograstic animals

ABSTRACT

The invention relates to a process for the preparation of dry lyophilized powdered formulations of probiotic bacteria for monogastric animal (for example poultry birds) using a combination of different polymers through the process of encapsulation. The resulting lyophilized powders of probiotic bacteria are having increased viability during animal feed processing, storage and upon transfer to the monogastric animal&#39;s digestive system.

FIELD OF THE INVENTION

The invention relates to a process for the preparation of drylyophilized powdered formulations of probiotic bacteria for monogastricanimal (for example poultry birds) using a combination of differentpolymers through the process of encapsulation. The resulting lyophilizedpowders of probiotic bacteria are having increased viability duringanimal feed processing, storage and upon transfer to the monogastricanimal's digestive system.

BACKGROUND

The monogastric animals have a simple gastrointestinal system consistingof oesophagus, stomach, small intestine, large intestine, colon andrectum assisted with liver and pancreas in some cases. The pH of thestomach is about 1.5-2.0 causing it to be a highly acidic environmentwherein survival of any microbe is a challenge. Post stomach the pHgradually changes from 2.0-7.0 in the small intestine whereas it becomesalkaline (8.5-9.0) in the colon. Generally in case of monogastricdigestive systems, the challenges for effective delivery of probioticbacteria are:

-   -   1. Acidic gastric environment affecting the viability and        adequate number of viable cells from reaching and colonizing the        small intestine which is the site of action.    -   2. Process difficulties especially heat treatment during feed        blending, pelletization, etc. and    -   3. Logistics and shelf life issues of probiotic with respect to        storage under differing conditions of temperature and moisture.

Since the probiotic bacteria are not tolerant to acidic environment instomach and get killed instantaneously at low pH, thus reducing theeffective probiotic microbe concentration required to enter the furtherparts of the system. Whether they are given through food or feed, theyface harsh processing conditions mainly that of heat. Heat toleranceneeds to be imparted as the majority of microbes cannot withstand highheat for extended time. With these technical hurdles, an additionalcommercial criterion is the need to survive exposure to harsh andvarying physical and environmental conditions of formulation storage.

A method for protecting the probiotic microbe from these adverseconditions is protection in the form of encapsulation. For developmentof the encapsulation technology, Bacillus amyloliquefaciens andPropionibacterium freudenrechii were selected as probiotic strains;however, the technology disclosed herein is equally applicable on otherorganisms.

Encapsulation is one known method to increase the shelf life ofprobiotic formulations. Encapsulation is a process in which tinydroplets or particles are wrapped with a protective coating yieldingcapsules enclosing the bacteria. Encapsulation of these probioticbacteria is generally used to maintain the viability during processingtill the site of action in GI tract. It is critical for the targeteddelivery of a minimum required cell count in GI tract. The probioticsare used with the animal feed, pharmaceutical products, and healthsupplements. They play a great role in maintaining gut health. As thesurvival of these bacteria in the digestive system is questionable so inorder to protect the viability of the probiotic bacteria, several typesof biopolymers such as alginate, chitosan, gelatin, whey proteinisolate, pectin, cellulose derivatives have been used for theencapsulation and several methods of encapsulation such as spray drying,extrusion, emulsion have been reported.

The invention presented herein discloses a process for the preparationof powdered formulations of probiotic bacteria on a large scale usingcombination of two different polymers resulting in a material matrixthat has a low porosity and that helps in lowering the exposure tounfavourable environmental conditions in the monogastric animal's gastrointestinal tract. These formulations are easy to handle and transportand more stable at ambient conditions.

DETAILED DESCRIPTION

In one embodiment of the invention, for development of the encapsulationtechnology, Bacillus amyloliquefaciens and Propionibacteriumfreudenrechii were selected on the basis of their known probioticproperties.

In another embodiment of the invention, about 20-hour old or freshlygrown probiotic cell biomass was used, unless otherwise stated.Different matrix polymers either individually or in combination wereevaluated in the microencapsulation process.

Non-encapsulated cells served as the control for in comparative cases.The total viable count for Propionibacterium freudenrechii in MRS mediumis about 2.98×10⁹ CFU/ml in static incubation and for Bacillusamyloliquefaciens 7.80×10⁹ CFU/ml under shaking incubation condition at37° C. for about 24 hours using conical flasks of 1000 mL capacity. Saidmedia is selected further for fermentation and for probiotic formulationdevelopment. Further said inoculum is inoculated in MRS broth andincubated at static condition for about 24 hours at about 37° C.Further, said broth is subjected to centrifugation with feed rate ofabout 500 ml/min to separate the wet cake. Next, the wet cake isimmediately removed aseptically in LAF unit (Laminar Air Flow). Furtherabout 0.5% carboxymethylcellulose (CMC) and about 1% of maltodextrin isused as an encapsulation agent. Such encapsulation solution issterilized at about 121° C. for about 20 minutes. Further said wet cakeand encapsulating chemicals (1% maltodextrin+0.5% CMC) are mixedproperly using vortex mixer to prepare a lyoslurry. Then said preparedlyoslurry is poured in trays and subjected to freezing at about −80° C.for about 2 hours. Post freezing, the frozen lyoslurry is subjected tolyophilization at about −50° C. for about 28 to about 36 hours toprepare lyopowder. The lyopowder obtained is analyzed for TVC and solidscontents. Said broth, wet cake, lyoslurry and lyopowder are seriallydiluted using sterile degassed saline Tween 80 solution and plated oversterile MRS agar plates. Said plates are incubated at about 37° C. forabout 48 to about 72 hours. The total solid and TVC values for broth,wet cake, lyopowder and for lyoslurry are enlisted in TABLE A and B.

TABLE A TVC of Bacillus amyloliquefaciens culture at different processstages No. Sample TS (% w/w) TVC (CFU/gm) 1 Harvest/Broth 2.71 1.7 ×10¹⁰ 2 Wet cake 14.86 2.6 × 10¹⁰ 3 Lyoslurry 6.52 4.1 × 10¹³ 4 Lyopowder97.5 1.8 × 10⁹ 

TABLE B TVC of Propionibacterium freudenreichii culture at differentprocess stages No. Sample TS (% w/w) TVC (CFU/gm) 1 Harvest/Broth 3.123.5 × 10¹⁰ 2 Wet cake 17.06 3.1 × 10¹² 3 Lyoslurry 9.00 3.4 × 10¹⁴ 4Lyopowder 96.1 2.3 × 10¹⁴

In another embodiment of the invention, said lyopowder formulations arepacked in air-tight containers and stored at room temperature at about50% relative humidity. The moisture contents of lyopowder formulationsare not more than 5% by weight. During storage after specified timeperiods the TVC analyses are carried out to check the viability ofbacteria in the formulations.

In another embodiment of the invention, before the production of drypowders of probiotic, the probiotic cultures were selected on the basisof survival efficiency with respect to simulated harsh environmentalconditions such as the exposure to high temperature and high bile saltsconcentration (gastric fluid) in stomach.

In another embodiment of the invention, the bacterial cultures wererevived from the glycerol stock using nutrient broth for Bacillus andMRS broth for Propionibacterium. For this 250 μl of culture fromglycerol stock was inoculated in 100 ml nutrient/MRS broth in 250 mlflask. The culture was incubated at 37° C., under shaking/staticcondition, for about 18±2 hours. From this 20 ml was taken in two 150 mlflask individually. One flask labeled as control flask, was diluted andplated on nutrient/MRS agar plate for total viable count (TVC) analysis.The second flask was treated at 90° C. for one minute in water bath.Dilution plating of the sample from both the flasks was done onnutrient/MRS agar medium for TVC determination. For dilution plating,0.9 ml of Tween-saline (0.05% Tween 80+0.5% sodium chloride) was addedin 2 ml Eppendorf tube, and 0.1 ml of culture was added to it to make10¹ dilutions and later serially diluted till 10¹². From each dilution0.1 ml was spread on nutrient agar and MRS agar plate respectively withthe help of sterile L-spreader. Plates were incubated at 37° C. for 24hours. Post incubation, growth was measured in terms of colony countsfor every dilution plated and TVC was calculated using formula:—

TVC=(No. of colonies×dilution factor)/aliquot plated

Percent survival for the culture after heat treatment was calculatedusing formula:

% survival=(TVC after heat treatment)/(TVC before heat treatment)×10.

In next embodiment of the invention, the probiotic cultures to be testedwere checked for tolerance to bile salts. Bile salt concentrationschosen for the assay were 0.5, 1.0, 1.5, 2 and 3%. The required amountof bile salt was mixed with nutrient (for Bacillus)/MRS broth (forPropionibacterium) and sterilized at 121° C. for 20 minutes. Thesterilized broth was inoculated using 1 ml freshly grown and centrifugedmedia free cell suspension of bacteria to be tested. This was incubatedat 37° C. for 24 hours under shaking/static condition. Growth wasmonitored visually and TVC was enumerated by dilution plating on agarmedium. To verify the tolerance the growth of the culture was alsotested on nutrient/MRS agar plate containing the required amount of bilesalt by surface spreading. In yet another embodiment of presentinvention, other polymers like 1% carboxy methyl cellulose sodium salt(CMC-Na), carboxy methyl cellulose sodium salt+maltodextrin (0.5% CMC+1%MD), 1% alginate, 1% kappa carrageenan, 1% gelatin and 1% pectin arealso tested alone or in combination as encapsulating agents forprobiotic bacteria for the preparation of the dry powders.

In yet another embodiment of present invention, the probiotic bacteriais one of Bacillus amyloliquefaciens or Bacillus licheniformis orBacillus subtilis or Bacillus coagulans or Propionibacteriumfreudenreichii or Pediococcus acidilactici combination thereof.

Embodiments provided above give wider utility of the invention withoutany limitations as to the variations that may be appreciated by theperson skilled in the art. A non-limiting summary of various embodimentsis given above, which demonstrate the advantages and novel aspects ofthe process disclosed herein.

Advantages:

-   -   1. A unique encapsulation process helps in restricted release of        probiotic microbe in the small intestine of monogastric        digestive systems using a combination of polymers for better        protection.    -   2. The probiotic cultures tested for encapsulation are already        tolerance tested for bile salts, thermal treatment, and survival        in simulated gastric and intestinal fluids.    -   3. Said encapsulation agents used have a low porosity thus        contributing to less exposure of the probiotic microbes to        environment.    -   4. The encapsulated dry formulations have increased bacterial        viability during processing and application in the monogastric        digestive system.    -   5. The probiotic dry product is more stable and easier for        handling and transport.

Examples provided below give wider utility of the invention without anylimitations as to the variations that may be appreciated by the personskilled in the art. A non-limiting summary of various experimentalresults is given in the examples, which demonstrate the advantages andnovel aspects of the process for preparation of dry probiotic microbeformulations using combination of polymers in a uniquemicroencapsulation technique.

Example 1

A strain of Propionibacterium freudenreichii (MTCC 1371) was used forthe preparation of dry powdered formulations. Said bacteria was grown indeMan Rogosa Sharpe (MRS) broth medium in static incubation condition atabout 37° C. for about 24 hours in conical flasks. The culture wasroutinely transferred and stored at about −80° C. in about 20% glycerolby volume between transfers. The total viable count in MRS medium wasabout 2.98×10⁹ CFU/ml.

A strain of Bacillus amyloliquefaciens (MTCC 10456) was used for thepreparation of dry powdered formulations. Said bacteria was grown innutrient broth or MRS broth medium in shaking incubation condition atabout 37° C. for about 24 hours in conical flasks. The culture wasroutinely transferred and stored at about −80° C. in about 20% glycerolby volume between transfers. The total viable count in MRS medium wasabout 7.8×10⁹ CFU/ml compared with the nutrient broth at about 5.7×10⁸CFU/ml.

Example 2: Thermotolerance & Bile Tolerance of the Formulted Bacteria

The bacterial cultures were revived from the glycerol stock usingnutrient broth for Bacillus and MRS broth for Propionibacterium. Forthis 2501 of culture from glycerol stock was inoculated in 100 mlnutrient/MRS broth in 250 ml flask. The culture was incubated at 37° C.,under shaking/static condition, for about 18 hours. From this 20 ml wastaken in two 150 ml flask individually. One flask labeled as controlflask, was diluted and plated on nutrient/MRS agar plate for totalviable count (TVC) analysis. The second flask was treated at 90° C. forone minute in water bath. Dilution plating of the sample from both theflasks was done on nutrient/MRS agar medium for TVC determination. Fordilution plating, 0.9 ml of Tween-saline (0.05% Tween 80+0.5% sodiumchloride) was added in 2 ml eppendorf tube, and 0.1 ml of culture wasadded to it to make 10¹ dilutions and later serially diluted till 10¹².From each dilution 0.1 ml was spread on nutrient agar or MRS agar platewith the help of sterile L-spreader. Plates were incubated at 37° C. forabout 24 hours. Post incubation, growth was measured in terms of colonycounts for every dilution and TVC was calculated. TABLE 3 shows thethermo tolerance of the bacteria in the formulation compared with plainculture.

TABLE 2 Heat exposure % Survival (90° C. for 1 BacillusPropionibacterium min) amyloliquefaciens freudenreichii Non 28.21% 2.34%encapsulated (original count - (original count - probiotic 7.80 × 10⁹,2.98 × 10⁹, count post count post heat treatment - heat treatment - 2.20x 10⁹ cfu/ml) 7.0 × 10⁷ cfu/ml) Encapsulated 86% 60% probiotic (originalcount - (original count - 6.90 × 10⁹, 1.62 × 10⁹, count post count postheat treatment - heat treatment - 5.93 × 10⁹ cfu/ml) 9.72 × 10⁸ cfu/ml)

Example 3

Here the probiotic bacteria were checked for tolerance to bile salts.Bile salt concentrations chosen for the assay were 0.5, 1.0, 1.5, 2 and3%. The required amount of bile salt was mixed with nutrient (forBacillus)/MRS broth (for Propionibacterium) and sterilized at 121° C.for 20 minutes. The sterilized broth was inoculated using 1 ml freshlygrown and centrifuged media free cell suspension of bacteria to betested. This was incubated at 37° C. for 24 hours under shaking/staticcondition. Growth was monitored visually and TVC was enumerated bydilution plating on agar medium. To verify the tolerance, the growth ofthe culture was also tested on nutrient/MRS agar plate containing therequired amount of bile salt by surface spreading. TABLE 3A and TABLE 3Bshow the data related to tolerance of bacteria upon encapsulation.

TABLE 3A Non-encapsulated Encapsulated Bile salt PropionibacteriumPropionibacterium Concentration freudenreichii freudenreichii (g %)(cfu/ml) (cfu/ml) 0  4.00 × 10¹²  2.9 × 10¹³ 0.5 3.80 × 10⁶ 3.5 × 10⁸1.0 4.20 × 10⁴ 3.1 × 10⁶ 1.5 3.90 × 10⁴ 2.6 × 10⁶ 2.0 3.30 × 10⁴ 4.9 ×10⁴ 3.0 2.40 × 10⁴ 1.19 × 10⁴ 

TABLE 3B Non-encapsulated Encapsulated Bile salt Bacillus BacillusConcentration amyloliquefaciens amyloliquefaciens (g %) (cfu/ml)(cfu/ml) 0 2.10 × 10¹⁵ 3.5 × 10¹⁴ 0.5 6.70 × 10¹³ 5.1 × 10¹³ 1.0 3.10 ×10¹² 1.1 × 10¹³ 1.5  1.0 × 10¹¹ 2.1 × 10¹¹ 2.0  2.8 × 10⁹ 3.1 × 10¹⁰ 3.0 1.6 × 10⁹ 1.8 × 10¹⁰

Example 4

Both Bacillus amyloliquefaciens and Propionibacterium freudenrechiicultures were inoculated in MRS broth flasks and incubated in staticcondition at about 37° C. for about 24 hours to form a starting culture.Next this starting culture was inoculated in 1.5 L MRS broth andincubated for about 24 hours at about 37° C. Post incubation both thebroths were subjected to centrifugation to separate the biomass.Polymers like 1% carboxymethylcellulose sodium salt (CMC-Na),carboxymethylcellulose sodium salt+maltodextrin (0.5% CMC+1% MD), 1%alginate, 1% kappa carrageenan, 1% gelatin and 1% pectin were tested asencapsulating agents for probiotic bacteria for the preparation of thedry powders. The stock solutions containing 1% concentration of theencapsulating agents were sterilized at about 121° C. for about 20 min.The biomass of both microbes obtained after centrifugation was mixedwith sterile solutions of encapsulation agents in 1:1 proportion. Saidmixture was mixed well using vortex mixer. Obtained lyoslurry was thensubjected to freezing at about −80° C. for 2 hours. Post freezing, thefrozen lyoslurry was subjected to lyophilization at about −50° C. forabout 28 to 36 hours to prepare the lyopowder [powdered probioticformulations]. Said lyopowder and lyoslurry were serially diluted usingsterile saline Tween 80 solution and plated over sterile MRS agarplates. Said plates were incubated at about 37° C. for about 24 to about72 hours according to the culture. Total viable counts analyses of saidlyopowder and lyoslurry were carried out to check the encapsulationpotential of polymers and loss of viability of probiotic cells afterlyophilization. The total viable counts of non encapsulated andencapsulated probiotic bacteria after lyophilization (lyopowder) aregiven in TABLE 4.

TABLE 4 ENCAPSULATION AGENTS SCREENING ASSAY TVC (CFU/gm) data forLYOPOWDER encapsulation materials used TVC 1% 0.5% CMC + 1% Sodium 1%Culture Identity CMC 1% MD Alginate Gelatin Propionibacterium 2.23 ×2.54 × 2.63 × 2.03 × freudenreichii 10¹² 10¹³ 10⁹ 10¹³ Bacillus 3.89 ×3.45 × 3.53 × 1.15 × amyloliquefaciens 10¹² 10¹⁴ 10⁸ 10¹⁰

The control (non-encapsulated) TVC for both cultures were

1. Propionibacterium freudenrechii: 1.16×10¹⁴ CFU/ml

2. Bacillus amyloliquefaciens: 1.10×10¹³ CFU/ml

Example 5: Assay for the Release of Encapsulated Cells in SimulatedGastric and Intestinal Fluids

To check the efficiency of release of the bacterial cells fromencapsulating materials at desired pH, the tolerance and release of thecells of Bacillus amyloliquefaciens and P. freudenrechii encapsulated in0.5% CMC+1% MD were studied in SGF [simulated gastric fluid] withrelease in SIF [simulated intestinal fluid]. The encapsulated bacteriawere resistant to the low pH of SGF and survived it for up to 6 hours.Similarly the encapsulated bacteria were easily released in SIF at highpH. The obtained data is shown in the TABLE 65A and TABLE 5B.

TABLE 5A ENCAPSULATION RELEASE EXPERIMENT IN SGF BacillusPropionibacterium amyloliquefaciens freudenreichii Time Non- Non- [h]encapsulated Encapsulated encapsulated Encapsulated 0 7.1 × 10⁶ 7.8 ×10⁷ 2.3 × 10⁵ 8.6 × 10⁷ 2 7.1 × 10⁶ 7.8 × 10⁶ 6.0 × 10⁴ 6.0 × 10⁷ 4 2.14× 10⁶  6.8 × 10⁶ 2.5 × 10⁴ 1.9 × 10⁷ 6 1.8 × 10⁶ 7.0 × 10⁶ 0 2.1 × 10⁷

TABLE 5B ENCAPSULATION RELEASE EXPERIMENT IN SIF BacillusPropionibacterium amyloliquefaciens freudenreichii Time Non- Non- [h]encapsulated Encapsulated encapsulated Encapsulated 0 1.1 × 10⁸ 6.5 ×10⁶  1.4 × 10¹⁰ 1.1 × 10¹¹ 2 3.5 × 10⁸ 5.9 × 10⁶ 2.17 × 10⁹  1.42 ×10¹¹  4 2.1 × 10⁸ 7.1 × 10⁶  9 × 10⁹ 3.3 × 10¹⁰ 6  4 × 10⁸ 6.9 × 10⁶ 2.3× 10⁹ 2.1 × 10¹⁰

Example 6: Analysis of Encapsulated Cells by Scanning ElectronMicroscopy

To establish the morphology of the particles of the dry lyophilizedpowders [formulations] of Bacillus amyloliquefaciens andPropionibacterium freudenreichii using the encapsulating agent [0.5%CMC+1% maltodextrin] as such and with the bacterial cells enclosed wereanalysed under a scanning electron microscope. The details of analysisare included below.

FIGS. 1 A & B shows the encapsulating agent as such as a powdery matter.FIGS. 1 C & D shows the encapsulating agent enclosing the bacterialcells of Bacillus amyloliquefaciens and Propionibacterium freudenreichiiforming particles of definite size and shape, respectively.

Example 7: Infrared Spectral Analyses of Encapsulated Formulations

To establish the spectral properties of the particles of the drylyophilized powders of Bacillus amyloliquefaciens and Propionibacteriumfreudenreichii with the encapsulation agent with or without bacteria,the samples were analysed by FT-IR spectrometry.

IR Spectroscopy: The culture inoculated into broth was incubated at 37°C. for 24 hr. The broth was centrifuged (8000 rpm, 10 min, 4° C.) tocollect the cells. The cells were washed three times with saline (0.85%sodium chloride). The cells were again re-suspended in saline andcentrifuged. The obtained cell pellet was pre-freezed at −80° C. for 2hr and lyophilized to obtain powdered form using lyophilizer. Allspectra were recorded using Spectrum100FT-IR spectrometer (PerkinElmer).At least 8 scans, between wave number region 4000 cm⁻¹ to 500 cm⁻¹ withspectral resolution of 4 cm⁻¹, were taken for average estimation.

FIG. 2A is the spectrum of carboxymethyl cellulose sodium salt as such.FIG. 2B is the spectrum of maltodextrin as such. FIG. 2C is the spectrumof 0.5% CMC+1% maltodextrin mixture without bacterial cells. FIG. 2D isthe spectrum of 0.5% CMC+1% maltodextrin mixture with bacterial cells.After mixing of both MD and CMC, absorption was not changed at majorfrequencies. However, after cell encapsulation, it exhibited newabsorptions at about 1150 cm⁻¹ and 1453 cm⁻¹ which are remarkable forthe encapsulated formulations of the bacteria herein in disclosed.

Embodiments provided above give wider utility of the invention withoutany limitations as to the variations that may be appreciated by theperson skilled in the art. A non-limiting summary of various embodimentsis given in the examples and tables, which demonstrate the advantageousand novel aspects of the process disclosed herein.

1. A process for the preparation of a dry lyophilized formulation ofprobiotic bacteria comprising steps of: (a) providing bile,thermo-tolerant cultures of probiotic bacteria; (b) subjecting saidculture to centrifugation to separate bacteria as a wet cake; (c) mixingsaid wet cake with an encapsulating agent forming a lyoslurry; (d)lyophilising said lyoslurry under desired conditions forming alyopowder; and (e) packaging said lyopowder in an air-tight container.2. The process of claim 1, wherein said probiotic bacteria is one ofBacillus amyloliquefaciens or Bacillus licheniformis or Bacillussubtilis or Bacillus coagulans or Propionibacterium freudenreichii orPediococcus acidilactici combination thereof.
 3. The process of claim 1,wherein said encapsulating agent is a mixture of carboxymethyl cellulosesodium salt and maltodextrin.
 4. The process of claim 1, wherein saidencapsulating agent comprises carboxymethyl cellulose about 0.5% byweight.
 5. The process of claim 1, wherein said encapsulating agentcomprises maltodextrin about 1% by weight.
 6. The process of claim 1,wherein said lyophilisation is performed at about −50° C. for about 30to about 36 hours.
 7. The process of claim 1, wherein said formulationis used as a component of monograstic animal feed supplement.
 8. Theprocess of claim 1, wherein said formulation is stable in SGF for up to6 hours.
 9. The process of claim 1, wherein said formulation releasesthe bacteria in SIF in less than 1 hour.
 10. The process of claim 1,wherein said formulation is tolerant to acidic conditions of the stomachof monograstic animals.